Systematic Review and Meta-Analysis of Congenital Toxoplasmosis Diagnosis: Advances and Challenges

Objective To understand how congenital toxoplasmosis (CT) diagnosis has evolved over the years, we performed a systematic review and meta-analysis to summarize the kind of analysis that has been employed for CT diagnosis. Methods PubMed and Lilacs databases were used in order to access the kind of analysis that has been employed for CT diagnosis in several samples. Our search combined the following combining terms: “congenital toxoplasmosis” or “gestational toxoplasmosis” and “diagnosis” and “blood,” “serum,” “amniotic fluid,” “placenta,” or “colostrum.” We extracted data on true positive, true negative, false positive, and false negative to generate pooled sensitivity, specificity, and diagnostic odds ratio (DOR). Random-effects models using MetaDTA were used for analysis. Results Sixty-five articles were included in the study aiming for comparisons (75.4%), diagnosis performance (52.3%), diagnosis improvement (32.3%), or to distinguish acute/chronic infection phases (36.9%). Amniotic fluid (AF) and placenta were used in 36.9% and 10.8% of articles, respectively, targeting parasites and/or T. gondii DNA. Blood was used in 86% of articles for enzymatic assays. Colostrum was used in one article to search for antibodies. In meta-analysis, PCR in AF showed the best performance for CT diagnosis based on the highest summary sensitivity (85.1%) and specificity (99.7%) added to lower magnitude heterogeneity. Conclusion Most of the assays being researched to diagnose CT are basically the same traditional approaches available for clinical purposes. The range in diagnostic performance and the challenges imposed by CT diagnosis indicate the need to better explore pregnancy samples in search of new possibilities for diagnostic tools. Exploring immunological markers and using bioinformatics tools and T. gondii recombinant antigens should address the research needed for a new generation of diagnostic tools to face these challenges.


Introduction
Congenital toxoplasmosis (CT) is a severe form of the disease caused by Toxoplasma gondii and occurs through the transplacental passage of tachyzoites from pregnant women to the fetus [1].Te risk of transmission depends on gestational age and clinical management for efective therapeutic intervention [2].Infected fetuses and newborns can sufer serious consequences of infection, such as retinochoroiditis, encephalitis, intracranial calcifcation, hydrocephalus, and death [3].
Procedures adopted to diagnose the infection, including test type/platform and antenatal period, vary according to the guidelines of each country/society.For example, in Brazil [70], CT is confrmed when a suspected case presents one of the following situations: presence of T. gondii DNA in AF, fetal tissue, or child body fuids; IgM or IgA and anti-T.gondii IgG reagent up to six months of life; serum levels of anti-T.gondii on the rise in at least two serial samples with a minimum interval of 3 weeks during the frst 12 months of life; anti-T.gondii IgG persistently reactive after 12 months of age; retinochoroiditis, hydrocephalus, or cerebral calcifcation (or associations between the signs) with reactive IgG.
Several countries have CT surveillance programs, but robust information on the frequency of CT transmission is limited to a few countries [71], so CT is substantially underestimated worldwide [72].Despite this, published data show that T. gondii is responsible for almost two-thirds of the estimated 1.9 million disability-adjusted life years (DALYs) [73], with an estimated 190,000 cases annually [74].Te incidence estimation of CT can be obtained from case report series, inferences from gestational toxoplasmosis, and testing babies at birth [71].Te disease is associated with fetal loss and neonatal death in approximately 3% of cases, [75] as well as craniocerebral/ocular sequelae [76].Subclinical disease at birth is present in 75% of cases, with symptoms that may start many years or even decades later [74].
To provide an understanding of the evolution of CT diagnosis over the years, we present here a review of methods that are currently employed for prenatal and postnatal CT diagnosis in several samples.It emphasizes the sample type, targets, and methods applied to diagnosis at diferent gestational ages using biological samples from pregnant women, fetuses, and newborns.Moreover, it brings insights into possible future challenges of CT diagnosis.

Search Strategy, Study Selection, and Data Extraction.
Our study followed the preferred reporting items for systematic reviews (PRISMA) guidelines [46].PubMed and Lilacs citation databases were searched from 2001 to 2020, combining the terms "congenital toxoplasmosis" or "gestational toxoplasmosis" and "diagnosis" and "blood" or "serum" or "amniotic fuid" or "placenta" or "colostrum."Only papers using human samples and written in English were included.
Te articles were selected by the Rayyan program, and seven authors conducted the preliminary selection based on abstracts and paper titles.After the frst selection, conficting decisions by at least three authors were considered for a second blind analysis.Afterward, articles considered eligible by at least four authors were included in the preliminary screening for full reading.Studies with at least one of the following criteria were excluded: studies evaluating exclusively infant samples, reviews or descriptive studies, articles with no eligible data, case reports, and studies approaching multiple infections.
Te following data were recorded from the selected studies: major goals, sample type, gestational age at sample collection, laboratory methods, and major results.For metaanalysis, data on molecular (PCR) and bioassay diagnosis performance were collected from articles that included these analyses and provided data about the number of samples, sensitivity, and specifcity.Only studies that reported the true positive, false negative, true negative, and false positive values or that these values could be calculated were included.Investigators collected data independently.When literature data interpretation was controversial, investigators discussed it and reached a consensus.Some studies considered essential to the review that were not included in any of the research bases were added to the introduction and discussion.

Statistical Analysis. Venn diagram was performed using
Bioinformatics and Evolutionary Genomics, available at https://bioinformatics.psb.ugent.be/webtools/Venn/.It was constructed to identify common and exclusive biological samples used in the selected studies.Meta-analysis of molecular diagnosis or bioassay in AF or placenta samples was performed using MetaDTA (version 2.0) [77,78], available at https://crsu.shinyapps.io/dta_ma/.Te diagnostic odds ratio (DOR), positive likelihood (LR+), and negative likelihood ratio (LR−) were used to determine the overall diagnostic accuracy.Sensitivity and specifcity points were shown along with forest plots and SROC curves.Te forest plots were edited using GraphPad Prism software.Heterogeneity and threshold efects were evaluated using the visual summary of SROC plots and random efects correlation, as described by Druce et al. [79].All summary parameters were calculated along with the associated 95% confdence interval (CI).

Analysis of the Included
Literature.Trough this systematic review, 1137 articles were found following the initial database search.In total, 517 articles were excluded from duplicate records, and 620 articles were screened based on title and abstract.From that, 523 articles were excluded, as it did not ft our flters.Te remaining 97 articles were evaluated by full reading, and 32 were excluded according to the criteria outlined in the Methods.Finally, 65 articles were included in the systematic review, and of these, 10 articles aiming for diagnostic performance on molecular assays (PCR) and/or bioassay were selected for meta-analysis.Details of the search and study selection procedures were described in a PRISMA fow diagram (Figure 1(a)).
To identify common and exclusive samples used in the selected studies, a Venn diagram was constructed (Figure 1(b)).Te analysis of samples used in the articles demonstrated that 9 studies were conducted employing exclusively AF, whereas 38 studies were performed exclusively with blood samples.Analyzing the articles with more than one sample, it was demonstrated that 10 articles used AF and blood, 2 articles used placenta and blood, 1 article used colostrum and blood, and 5 articles used AF, blood, and placenta simultaneously (Figure 1(b)).

Diagnostic Methods Tat Employed Amniotic Fluid
Samples.From the careful selection, 24 articles from the total used AF as a sample (Table 2).Regarding gestational age on sample collection, 5 articles collected AF between the 14th and 26th gestational weeks (GW), 9 articles collected AF between the 14th and 41st GW, and 5 articles collected additional samples at birth.Ten articles did not provide details on the date of sample collection.
All selected articles used parasites and/or T. gondii DNA as targets of study.Concerning the assays performed in those studies, all selected articles performed PCR and 8 of them also performed bioassay by mouse inoculation.Te B1 gene was the most commonly used gene in PCR (19/24 articles).Twelve articles (12/24 articles) used other genes such as 529bp, RE-sequence, P30, and others.Two articles (2/24 articles) did not provide details about PCR.Most studies using bioassay did not provide information about the methodology employed.

Diagnostic Methods
Tat Employed Blood Sample.Of the total, 56 articles used blood samples for CT diagnosis (Table 3).Peripheral blood samples were collected from pregnant women (M-PB) (51 articles/78.4%),cord blood by cordocentesis (P-CB) (2 articles), cord blood at the time of delivery (N-CB) (11 articles), or/and neonatal peripheral blood (N-PB) (18 articles).
When the target was examined, 53 of 56 articles analyzed antibodies against T. gondii by immunoassays.4).Placentas were used to search for parasites and/or T. gondii DNA by PCR or bioassay.PCR and bioassay were performed in 4 studies, whereas 2 articles used exclusively PCR and 1 article used exclusively bioassay.B1 was the most commonly used gene for PCR in placentas.REP529 and RE-sequence were applied in the PCR in 2 and 1 articles, respectively.Concerning the bioassay, 2 articles employed Swiss females and 2 other articles did not report details about this methodology.Colostrum was collected up to 3 days after birth.Samples were analyzed to detect anti-T.gondii antibodies using ELISA and western blot immunoassays.

Discussion
To the best of our knowledge, the frst conclusive reported case of toxoplasmosis in newborns was diagnosed based on encephalomyelitis and chorioretinitis fndings in infant postmortem tissues.Mice and rabbit tissue inoculations evidenced an infection with protozoa morphologically compatible with T. gondii.[80] Afterward, a dye test was developed to evaluate the presence of the specifc antibody [81].Following, a description of T. gondii isolation from the placenta gave a new puzzle connection about congenital infection [82].Later, a 15-year prospective study brought important information about CT diagnosis [83].
Te present study aimed to investigate commonly used diagnostic methods for CT and understand the accuracy of these methodologies.From these data, we seek out new diagnostic proposals that can be investigated, bringing insights into new diagnostic approaches.Our data suggested that, in the last 20 years, the samples and assays used for CT    Journal of Tropical Medicine Journal of Tropical Medicine Journal of Tropical Medicine  - [6] dnr: details not reported; ELISA: enzyme-linked immunosorbent assay; GW: gestational weeks, CT: congenital toxoplasmosis; PCR: polymerase chain reaction; qPCR: quantitative PCR; SNPs: single nucleotide polymorphism; SSP: sequence-specifc primers' amplifcations.Journal of Tropical Medicine     diagnosis are basically the same as those of past decades.Few studies evaluated the efectiveness of alternative samples, such as colostrum.Te majority of the studies in this review used blood samples mainly for serological screening, and a few studies used more than one type of sample for diagnostic investigation.Te increase in amount and time of sample collection represents a gain for CT diagnosis that has also evolved in accuracy [30].A schematic model representing the types of samples and methods used for T. gondii detection before and after birth is shown in Figure 3. Techniques used for CT diagnosis have advanced; however, many difculties are still encountered in screening pregnant women and fetuses.Our review suggested that one of the major challenges of CT diagnosis is dating the T. gondii infection [30].T. gondii-specifc immunoglobulin (IgG and IgM) searches are often used to investigate when the infection occurred [3,25,30].IgG-avidity helps determine the risk of T. gondii transmission at any time during pregnancy [29].Conversely, avidity assay results classifed as borderline or low can be erroneously interpreted as consistent with a recently acquired infection [25].

Journal of Tropical Medicine
Many studies in this review aimed to distinguish between acute/chronic infection phases.However, few of them used new approaches, such as evaluating more options for T. gondii antigens for the improvement of enzyme assays [26,33,39,44,47] or employing bioinformatics tools, such as epitope prediction for CT diagnosis [56].Te use of specifc molecular markers is a promising option in T. gondii serodiagnosis and can be useful for dating the infection.Recombinant proteins are highly advantageous for improving the diagnostic assay.Te combination of several recombinant antigens with multiple immunodominant  epitopes signifcantly increases the probability of detecting specifc antibodies at diferent stages of the infection [4,84].Besides, avidity assays based on recombinant antigens have potential clinical usefulness for diagnosing the acute phase of T. gondii infection [26].Many articles in this review assessed performance diagnosis or combined methods to evaluate performance improvement.Te importance of diagnostic accuracy should be emphasized in order to conduct the correct treatment to avoid transplacental transmission and to prevent unnecessary and potentially toxic treatment or termination of pregnancy [5].A combination of methods can also improve diagnostic accuracy [85].T. gondii detection by DNA amplifcation or parasite isolation is complementary to serological tests.Tese methods are particularly important in AF to indicate fetal infection [86].
Our meta-analysis of diagnostic performance for DNA/ parasite detection (PCR and bioassay) demonstrated a variation of sensitivity values.Although amniocentesis is a highly invasive method, amniotic fuid was the sample that presented the best values of PCR sensitivity.Likewise, detecting parasite burden in AF helps predict the severity of clinical symptoms in neonates congenitally infected [72].Te antenatal diagnosis of CT is the greatest advance in the cases of fetal infection, and the use of PCR analysis of AF is the most commonly used and accepted laboratory method for CT diagnosis during gestation [86].Normally, negative PCR in AF indicates the absence of fetal infection, although it cannot be ruled out completely.However, a positive PCR result almost certainly indicates a congenital infection [14].
Variation in sensitivity can be associated with dissimilarity in the PCR methodologies, time of sample collection, time of maternal seroconversion, infuence of treatment [19], and disparity in performance among laboratories.A considerable number of PCR results show the absence of T. gondii DNA amplifcation concomitant with CT, indicating low sensitivity [13].In some cases, these results can be explained by the absence of optimal amniocentesis at the time of sample collection [23].It can also be attributed to the inefciency of parasite DNA extraction and amplifcation, mainly due to the low concentrations of tachyzoites in the AF collected [87].
Some studies in this review implemented comparisons of the sensitivities of PCR methodologies, including comparisons between target genes.By far, B1 is the most employed gene in PCR followed by REP529.Tere is an important discrepancy in the literature concerning the best target gene for PCRs.Some studies in this review indicated that REP529 is more sensitive compared to B1 [18,68].Another study showed no discordance between these two targets [88].Tese results drive the need for more studies comparing target genes.
Te present study aimed to estimate all possible random efects for CT diagnosis data and compare them without applying alternative simplifcations [89].Random efect correlations of +1 or −1 found for bioassay indicated a hit/ truncation on model parameters.Tere were no explicit convergence problems, yet a few studies and/or sparse data (e.g., indicating no/low heterogeneity in the specifcity parameter) are possibly data problems.In this situation, the power of the model can be compromised [90] and the diagnostic parameters should be interpreted with caution.
Our study to review pertinent publications and assess diagnostic test accuracy performance for detecting T. gondii infection was defned by the limited number of available studies that meet the selection criteria.Insufcient reporting regarding population characteristics/recruitment and data about sample number, sensitivity, and specifcity was an issue in many studies, with information often provided with little detail.Our results also indicate the need for searching for new diagnosis methodologies, improving existing techniques, and providing proper training for professionals involved in the routine diagnosis of CT [91].Moreover, since higher T. gondii concentrations in AF are correlated with clinical signs in neonates, quantitative PCR can be important to evaluate the prognosis of the fetal infection [92].Another important methodology that was scarcely used in selected articles, but is of great relevance, is T. gondii isolation followed by genotyping.Tis technique allows the identifcation of nonclonal strains.Dubey and coworkers [93] identifed 58 diferent T. gondii genotypes circulating in Brazil.Studies identifed a new T. gondii strain in southern Brazil that was strongly related to the toxoplasmosis outbreak in 2018 [94].Such studies allow the surveillance of new circulating genotypes that are usually related to more severe forms of toxoplasmosis.
Variation in performance in diagnostics also drives the need for new diagnostic approaches, and few studies have focused on this aim.Tere is a potential for using biomolecules present in AF for complementary diagnosis.Using complementary biomarkers, such as immune response mediators, could help endorse and increase the reliability of diagnosis in AF.Previous studies suggested the ability to use cytokines, such as TGF-β in AF, as biomarkers to predict acute T. gondii infection [95].Another possibility is the use of AF cellularity as a potential biomarker of congenital infection.Studies using AF from the second and third trimester of pregnancy highlighted the cell dynamics in this compartment [96].Infammation, whether associated or not with infection, causes an increase in the number of immune cells.Some studies in this review collected samples at birth, including AF, placenta, and blood.Samples collected at the time of delivery are especially signifcant in the absence of prenatal follow-up, making it possible to anticipate diagnosis and treatment for newborns.T. gondii isolation from placenta is a useful tool to study CT and is an easily available sample.Placental analysis can be important to diagnose infection when AF is either not positive or not analyzed.Besides, placental samples can be useful for isolation and genotyping of the parasite, especially in outbreaks [97].However, it is important to highlight that maternal treatment can infuence the efcacy of placental analysis since T. gondii was less frequently isolated in the placenta of treated women [62,64,66].
Postnatal follow-up, based on after-birth samples, remains necessary in the frst year of life to fully exclude the infection when PCR or serological results were negative [29,86].Diagnosis based on cell immunity has been increasingly used as a complementary diagnostic to monitoring infants [98,99].Tis potential methodology should also be explored for maternal samples during prenatal follow-up.Alternative biological samples, such as colostrum from puerperal women [69] and saliva [44,100], also provide interesting data on humoral immunity and promising results for diagnosing toxoplasmosis using noninvasive sample collection.Searching for IgG and IgG-subclasses produced by newborns compared with maternal antibody responses [32] can also be promising for CT diagnosis.
Preventive and diagnostic measures for pregnant women vary between countries.Although prenatal diagnosis of CT is available, there is no international framework for monitoring the disease, and it is a neglected disease in most countries [1].Te absence or incomplete prenatal screening and treatment have been identifed as an important risk factor for CT [87].Tus, the screening and prevention measures against toxoplasmosis should be made mandatory for pregnant women attending the antenatal clinic [4].

Conclusion
In conclusion, this review points out that the assays employed in the research are basically the same traditional approaches available for clinical purposes.Tese assays showed important variations in diagnostic performance that can result in undiagnosed CT.Tese results challenge us to search for new generations of diagnostic tools and improve existing techniques, together with eforts towards increasing the feasibility of laboratory testing.

Figure 1 :
Figure 1: Details of search and study selection procedure.(a) Te PRISMA fow diagram describing the study design process.(b) Venn diagram constructed to identify common and exclusive samples used in the included studies.

Figure 3 :
Figure 3: Schematic model represents the types of samples and methods used to T. gondii detection before and after birth.Maternal peripheral blood (M-PB), prenatal cord blood (P-CB), and amniotic fuid (AF) are the samples collected for gestational and congenital toxoplasmosis diagnosis before birth.After birth, the samples that can be collected are maternal peripheral blood (M-PB), colostrum (CL), placenta (PL), amniotic fuid (AF), neonatal cord blood (N-CB), and neonatal peripheral blood (N-PB) to confrm the congenital toxoplasmosis.Te target and methods most used to detect the infection are Toxoplasma DNA by PCR; the parasite by bioassay (culture and/ or mouse inoculation); and antibodies anti-Toxoplasma by immunoassay.PCR: polymerase chain reaction.Figure created using images from Servier medical art by Servier licensed under creative commons attribution 3.0 France (CC BY 3.0 FR).
and Table5.Te RE correlation for the bioassay

Table 1 :
Major goals of selected articles.

Table 3 :
Diagnosis methods in blood samples.

Table 4 :
Diagnosis methods in postnatal samples.
Forest plot of the sensitivity and specifcity estimates and 95% confdence intervals (CI) for PCR and bioassay in amniotic fuid or placenta samples according to the single study sets.Estimates of sensitivity and specifcity from each study are shown as solid yellow squares for PCR in amniotic fuid, solid yellow circle for bioassay in amniotic fuid, solid purple squares for PCR in placenta, and solid purple circle for bioassay in placenta.

Table 5 :
Summary estimates of diagnostic accuracy of molecular and bioassay techniques for the diagnosis of T. gondii infection.